FIG. 3.
DEF-1 is an SH3 binding protein. (A) Lysates made with NP-40 lysis buffer from NIH 3T3 cells expressing pLNSL7 alone (pLN) (38) or pLNSL7/HA-tagged DEF-1 (pLN/DEF-1 HA) were passed over the noted columns. Bound proteins were immunoblotted with an anti-HA antibody. (B) Bovine brain (brain extract) or Sf9 cells infected with a pp60c-src baculovirus (Bv Src) was lysed with NP-40 lysis buffer and passed over a column consisting of GST alone, GST-DEF-1-C1 (amino acids 777 to 926; SH3 binding sites), or GST-DEF-1-C2+3 (amino acids 928 to 1129; proline-rich repeat plus SH3 domain). Bound proteins were immunoblotted with an anti-pp60c-src antibody. (C) Bovine brain extracts were passed over a GST-DEF-1-C1, C2 (amino acids 934 to 1036; proline-rich repeat) or C3 column (amino acids 1074 to 1129; SH3 domain), and bound proteins were blotted for DEF-1 by using an anti-DEF-1 rabbit polyclonal serum. The brain lysate was immunoprecipitated with preimmune (pre-imm.) or anti-DEF-1 serum (αDEF-1) or passed over a column of GST alone (GST) as a control.